Many non-coding RNAs are processed and assembled into mature RNPs in a complex, multistep process involving endonucleolytic cleavage, exonucleolytic cleavage, nucleotide modification, proper folding of the RNA, and ordered assembly with proteins. S. cerevisiae Rntlp is a key enzyme in the processing of rRNAs, snRNA, snoRNAs, and in degradation pathways for some pre-mRNAs and mRNA. In addition to these activities, RNase Ills in higher organisms have been found to play key roles in miRNA and siRNA processing, as members of the Dicer and Drosha family of enzymes. Rnt1 p substrates include some H/ACA snoRNPs, a class of RNPs that are involved in modification of specific uridines to pseudouridines in rRNA. Among the proteins that are essential for H/ACA snoRNP biogenesis is Shql In this grant, we propose to investigate the roles of Rntlp and Shq1 in processing and assembly of RNPs using a structure based (NMR and crystallography) approach. The specific aims are: (1) Investigate the interaction of Rntlp dsRBD with different substrates. To probe the structural basis of target site specificity by Rntlp dsRBD, we will study the structures of RNA substrates with different loop and stem sequences and their interaction with Rntlp dsRBD. (2) Characterize the interaction of Rntlp dsRBD with non-substrate dsRNA. We will investigate the structure and binding kinetics of non-specific complexes of the Rnt1 p dsRBD with RNA, in order to provide insight into the contributions of the second dsRBD to complex formation and into how Rntlp scans the RNA to identify target binding sites. (3) Determine how the Rntlp EndoND is oriented relative to the dsRBDs when bound to substrate RNA and investigate the structure and function of the N-terminal domain. (4) Investigate the structure and function of Shqlp and interactions with Naflp, Nhp2p, and Rntlp. We will structurally characterize the domains of Shq1 and its interactions with other proteins, and combine this information with mutational analysis to help define the role of Shq1 in H/ACA snoRNP biogenesis. Lay: It is estimated that more than 90% of RNA in humans that is translated from DNA is non-coding for proteins. This RNA is involved in virtually all aspects of gene regulation and cell physiology, and errors in the correct processing, modification, and assembly of RNA can lead to a variety of diseases in humans. Our structural studies will provide molecular insight into how non-coding RNAs are regulated for cell function. [unreadable] [unreadable] [unreadable]